In human lupus nephritis, the severity of tubulointerstitial inflammation on biopsy correlates with the risk of subsequent progression to renal failure 1. Tubulointerstitial inflammation, and not glomerular inflammation, is associated with in situ clonal selection 2 and expansion of B cells reactive with antigens associated with inflammation. Certainly antigen recognition by the B-cell antigen receptor provides signals necessary for B-cell selection. However, additional second signals, derived from antigen-specific T cells or pattern recognition receptors, are required for B-cell proliferation. Therefore, we hypothesized that in situ B cells were receiving help from in situ T cells.

Consistent with our hypothesis, using four and five color confocal microscopy we observed CD4⁺ICOS⁺PD-1⁺ T cells in close apposition with CD20⁺ B cells. To quantitate the relationship between these presumptive T_FH cells and B cells, we developed novel, automated and therefore unbiased algorithms (using Python/Linux) to define the location of cells expressing multiple surface markers and then to assess the shortest distances between the same or different cell types. These analyses revealed that B cells or T_FH cells were never in close approximation with each other. In contrast, an average of 42% of T_FH cells in renal biopsies from lupus patients were in very close contact with B cells. Three-dimensional imagining of these conjugates revealed that closely opposed T_FH and B cells had formed atypical supramolecular activation complexes containing polarized TCR, LFA-1, MHC class II and ICAM-1 molecules. This suggests that the T_FH and B cells in these conjugates are antigenically related. The difference between the observed T:B conjugate rate, and that predicted to arise by chance, was highly significant at up to P = 10^-42. Finally, qPCR of mRNA captured from in situ ICOS⁺ T cells revealed that they had expression profiles consistent with them being bona fide T_FH cells.

These data demonstrate that T_FH cells are probably contributing to in situ humoral immune responses in lupus nephritis. Furthermore, our results indicate that quantitative imaging can be used to reveal important cell:cell interactions in human tissue.